During the last fiscal year, the Skeletal Biology Section reports activities in all three areas. 1) Biological activity of stem cells &#8232;&#8232;&#8232; &#8232; BMSC populations are able to recreate a bone/marrow organ in vivo due to the presence of the multipotent SSC subset. However, it is known by clonal analysis that not all clonogenic BMSCs are multipotent. In order to determine differences between clonogenic multipotent SSCs and similarly clonogenic but non-multipotent BMSCs, we established single colony-derived strains (SCDSs, initiated by individual Colony Forming Unit-Fibroblasts) and determined their differentiation capacity by vivo transplantation. In this series of human SCDSs (N = 24), 20.8% formed fibrous tissue (F), 66.7% formed bone (B), and 12.5% formed a bone/marrow organ, and thus were multipotent (M). RNA isolated from 12 SCDSs just prior to transplantation was analyzed by microarray. Although highly similar, there was variability from one SCDS to another, and SCDSs did not strictly segregate into the three functional groups (F, B or M) by unsupervised hierarchical clustering. We then compared 3 F-SCDSs to 3 M-SCDSs that did segregate. Genes associated with skeletogenesis, osteoblastogeneis, hematopoiesis, and extracellular matrix were over-represented in M-SCDSs compared with F-SCDSs. These results highlight the heterogeneity of SSCs/BMSCs, even between functionally similar SCDSs, but also indicate that differences can be detected that may shed light on the character of the SSC. 2) BMSCs/SSCs in disease&#8232;&#8232;&#8232;&#8232; Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD) are caused by germline mutations in genes that control telomere length, leading to very short telomeres and limited proliferative potential of hematopoietic stem cells (hence, bone marrow failutre). We found that skeletal stem cells (SSCs) within the BMSC population may contribute to the hematologic phenotype of TBD individuals. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to bone marrow failure in TBD. 3) Stem cells in tissue engineering and regenerative medicine In current orthopaedic practice, there is a need to increase the ability to reconstruct large segments of bone lost due to trauma, resection of tumors and skeletal deformities, or when normal regenerative processes have failed such as in non-unions and avascular necrosis. BMSCs, when used in conjunction with appropriate carriers, represent a means by which to achieve bone regeneration in such cases. While much has been done at the bench and in pre-clinical studies, moving towards clinical application requires the generation of clinical grade cells. We and our collaborators in the Cell Processing Section, DTM, NIH Clinical Center have developed an FDA-approved cell manufacturing procedure for the ex vivo expansion of high quality, biologically active human BMSCs. Bone marrow aspirates (8 mls) are analyzed for quality (colony forming efficiency and amount of peripheral blood). The adherent BMSCs in the aspirates are first expanded in T75 flasks with standard nutrient medium containing 20% fetal bovine serum until 80% confluency, and subsequently expanded in multilayer cell factories for a total of three passages. Sterility and viability testing are performed at each passage. Between 1.5 2.0 x 109 cells per donor can be generated. The cells are negative for hematopoietic and endothelial markers, and positive for stromal cell markers (CD146, CD 105, CD90, CD73, and CD29), demonstrating their identity and purity. Importantly, the manufactured cells are able to form a bone/marrow organ upon in vivo transplantation into immunocompromised mice, indicating that scale up did not reduce their essential biological functions (potency). Additional testing also determined their stability after long-term storage (liquid nitrogen), and at different time points from harvest at room temperature and at 4 C. Based on the literature, the BMSC confluence criteria used to determine passage and harvest timing vary widely from center to center, and the impact of confluence on BMSC properties remains controversial. We aimed to examine the effects of confluence on BMSC properties and confluence-associated markers were identified. BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony forming efficiency, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed. Confluence-dependent changes were detected in the expression of several cell surface markers, and 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. In addition, changes in lactate and glucose levels correlated with BMSC density. BMSC characteristics change as confluence increases. Based on the analyses performed, 100% confluent BMSCs may have compromised pro-angiogenesis properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry expression may be used to confirm that the BMSCs have been harvested at the appropriate confluence.